Nitrogen Loss from Pristine Carbonate-Rock Aquifers of the Hainich Critical Zone Exploratory (Germany) Is Primarily Driven by Chemolithoautotrophic Anammox Processes.

Swatantar Kumar,Kirsten Küsel,Patricia Geesink,Bo Thamdrup,Kai-Uwe Totsche,Valérie F. Schwab,Susan E. Trumbore,Martina Herrmann

doi:10.3389/fmicb.2017.01951

Abstract

Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5–4.7 nmol L−1 d−1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L−1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification.

Highlights

Over the last decades, human impact on the nitrogen cycle has resulted in increasing concentrations of nitrate in groundwater, which is of growing concern on a global scale (Galloway, 2005; Burgin and Hamilton, 2007; Schlesinger, 2009)
Nitrate concentrations are higher in the HTL wells compared to the HTU wells except well H32, which shows the highest nitrate concentration, while ammonium concentrations are higher in HTU compared to the wells of HTL with highest concentrations of 30 μmol L−1 in the groundwater of well H53 (Figure 1)
Along with the high representation of anammox-bacteria based on 16S rRNA-targeted Illumina sequencing, maximum abundances of hzsA genes, and two orders of magnitude higher hzsA compared to nirS gene transcripts, these findings provided strong support for these wells being an anammox hotspot within the heterogeneous carbonate-rock aquifer system of the Hainich Critical Zone Exploratory (CZE)

Summary

Introduction

Human impact on the nitrogen cycle has resulted in increasing concentrations of nitrate in groundwater, which is of growing concern on a global scale (Galloway, 2005; Burgin and Hamilton, 2007; Schlesinger, 2009). Especially under conditions of organic carbon limitation as they may occur in pristine limestone aquifers, autotrophic nitrate reducing processes such as autotrophic denitrification or anaerobic oxidation of ammonium (anammox) are likely to become more competitive. Subsequent studies suggested that anammox could be an important process responsible for nitrogen loss from ammonium- and nitrate-contaminated groundwater with up to 90% of nitrogen loss being attributed to anammox (Clark et al, 2008; Moore et al, 2011; Robertson et al, 2012; Hanson and Madsen, 2015; Smith et al, 2015)

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